The long-term objective of this proposal is to determine the mechanisms by which histone H2AX controls cellular responses to double strand breaks (DSB) and how it functions in double strand break repair (DSBR). H2AX undergoes phosphorylation on serine 139 of its C terminal tail in response to DNA damage. Mice lacking H2AX exhibit genomic instability and cancer predisposition. We recently developed a novel reporter for analysis of sister chromatid recombination (SCR), a major homologous recombination (HR) pathway in somatic cells. We found that H2AX serine 139 controls HR, including SCR. Remarkably, this function of H2AX appears to be conserved across evolution. Further, we found that H2AX regulates the "choice" between distinct DSBR pathways, favoring sister chromatid recombination (SCR) and suppressing single strand annealing (SSA). Other work suggests a role for H2AX in the third major DSBR pathway, non-homolgous endjoining (NHEJ). We believe that structural elements of H2AX in addition to serine 139 likely contribute to H2AX recombination functions. To test this hypothesis, we will assess quantitatively the role of individual residues of H2AX in HR/SCR, SSA and NHEJ (Aim 1). A number of DNA damage responsive protein complexes are recruited to chromatin following H2AX phosphorylation. Some of these may contribute to H2AX-dependent recombination functions. We will test this hypothesis by identifying new H2AX interaction partners and by studying the function of these and other known H2AX interactors in regulation of DSBR, including HR/SCR, SSA and NHEJ. We will attempt to examine dynamic aspects of the H2AX response by measuring the recruitment of repair factors to the site of a DSB in H2AX+/+ vs. H2AX-/- isogenic primary cells (Aim 2). Defects in recombination frequently cause increased mutation rates in other genes. We will assess the mutagenic consequences of H2AX dysfunction (Aim 3). This work will therefore significantly advance our understanding of how H2AX acts as a tumor suppressor gene. [unreadable] [unreadable]